The present invention relates to pharmaceutical compositions comprising a peptide derived from fibroblast growth factor as an active ingredient.
Some Fibroblast Growth Factors (called FGF hereinafter) are known to have various physiological activities, and a variety of studies have been made with the expectation of their use as pharmaceuticals. A food intake inhibitory effect is known to be one of the physiological activities of FGF. The FGF that is released from ependymocytes of the cerebral ventricle wall by responding to a glucose concentration in the brain, is considered to act on neurons of the feeding center in the brain repressively, resulting in the food intake inhibitory and weight-reducing effects.
Since FGF is a protein comprising one hundred and several tens of amino acids, however, there were the following problems with its use, as is, as a pharmaceutical: 1) the synthesis of FGF is difficult; 2) immunological rejection occurs in vivo; 3) administration methods are limited; 4) it is difficult to conserve FGF without causing changes of the active three-dimensional structure.
Therefore, there are strong demands on development of medicaments comprising FGF with physiological activity, particularly, food intake inhibitory effect, without any of the above problems.
An object of the present invention is to provide a pharmaceutical composition having physiological activity, particularly, food intake inhibitory effect of FGF and having no problems that occurs when FGF is used directly as a medicament.
The inventors have studied extensively and intensively to solve above problems and have now found that a partial peptide derivative, Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Val-Ala-Ala-Ala-Asn-Try-Lys-Lys-Pro-Lys-Leu (SEQ ID NO:3) (Lin Y. Z. et al., J. Biol. Chem., 270, 14255-14258 (1995), of an FGF previously known to have superior cell membrane transfer and cell nuclear membrane transfer, unexpectedly has strong food and water intake inhibitory effects. The inventors have further found based on this finding that the peptide which is obtained by combining a peptide with cell membrane transfer and a peptide with cell nuclear localization has strong food and water intake inhibitory effects, and accompanying weight-reducing and obesity-inhibiting effects, thereby completing the present invention.
Thus, the present invention comprises the following inventions:
(1) A pharmaceutical composition comprising a peptide represented by the following formula (I):
MTS-X-NLSxe2x80x83xe2x80x83(I)
xe2x80x83wherein MTS represents a membrane-transfer amino acid sequence; X represents a direct bond or a linker sequence; and NLS represents a nuclear localization amino acid sequence, or a pharmaceutically acceptable salt thereof as an active ingredient.
(2) The pharmaceutical composition of (1) above, wherein the MTS is represented by the following amino acid sequence:
(a) an amino acid sequence of SEQ ID NO:1; or
(b) an amino acid sequence comprising deletions, substitutions or additions of one or more amino acid residues relative to the amino acid sequence of SEQ ID NO: 1.
(3) The pharmaceutical composition of (1) or (2) above, wherein the NLS is represented by the following amino acid sequence:
(a) an amino acid sequence of SEQ ID NO:2; or
(b) an amino acid sequence comprising deletions, substitutions or additions of one or more amino acid residues relative to the amino acid sequence of SEQ ID NO:2.
(4) The pharmaceutical composition of any one of (1) to (3) above, wherein the pharmaceutical composition is for inhibition of food intake.
(5) The pharmaceutical composition of any one of (1) to (3) above, wherein the pharmaceutical composition is for inhibition of water intake.
(6) The pharmaceutical composition of (4) above, having a weight-reducing and/or obesity-inhibiting effect.
(7) The pharmaceutical composition of (5) above, having a weight-reducing and/or obesity-inhibiting effect.
The present invention will be described in detail as follows.
A pharmaceutical composition of the present invention is characterized in that it comprises, as the active ingredient, a peptide represented by the following formula (I):
MTS-X-NLSxe2x80x83xe2x80x83(I)
where MTS represents a membrane-transfer amino acid sequence; X represents a direct bond or a linker sequence; and NLS represents a nuclear localization amino acid sequence, or a pharmaceutically acceptable salt thereof.
As used herein, the term xe2x80x9cmembrane-transfer amino acid sequencexe2x80x9d represented by MTS means an amino acid sequence corresponding to a peptide with cell membrane transfer properties, and the term xe2x80x9cnuclear localization amino acid sequencexe2x80x9d represented by NLS means an amino acid sequence corresponding to a peptide with nuclear localization properties.
MTS may be any amino acid sequence as long as it is a membrane-transfer amino acid sequence, but particularly preferred is the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence comprising deletions, substitutions or additions of one or more amino acid residues relative to the amino acid sequence of SEQ ID NO: 1.
As used herein, the term xe2x80x9cone or more amino acid residuesxe2x80x9d does not limit the number thereof so far as the cell membrane transfer of a peptide corresponding to the amino acid sequence is not lost.
The number of amino acid residues is, but not limited to, normally 7-40, preferably 16.
The NLS may be any amino acid sequence as long as it is a nuclear localization amino acid sequence, but particularly preferred is the amino acid sequence according to SEQ ID NO:2 or an amino acid sequence comprising deletions, substitutions or additions of one or more amino acid residues relative to the amino acid sequence of SEQ ID NO:2.
As used herein, the term xe2x80x9cone or more amino acid residuesxe2x80x9d does not limit the number so long as the nuclear localization of a peptide corresponding to the amino acid sequence is not lost.
The number of amino acid residues is, but not limited to, normally 3-15, preferably 4-7.
In the formula (I), X represents a direct bond or a linker sequence. MTS and NLS may be bound together directly, or otherwise, a linker sequence may intervene between MTS and NLS. The linker sequence includes, for example, amino acid sequences comprising one or more Ala or Gly residues, or combination thereof. The number of amino acid residues in the linker sequence is, but not limited to, normally 1-20, preferably 2-7.
The peptide represented by the formula (I) includes for example peptides in which MTS is located on the N-terminal side while NLS on the C-terminal side, and peptides in which MTS is located on C-terminal side while NLS on the N-terminal side.
The number of the amino acid residues represented by the formula (I) is, but not limited to, normally 10-40, preferably 23-27.
The peptides represented by the formula (I) can be chemically synthesized, for example, by known peptide synthesis methods.
Examples of peptide synthesis methods include azide, acid chloride, acid anhydride, mixed acid anhydride, DCC, activated ester, carboimidazole, oxidation-reduction, and DCC-additive (HONB,HOBt,HOSu) methods. (Schreder and Luhke xe2x80x9cThe Peptidexe2x80x9d Vol.1 (1996), Academic Press, N.Y., USA; Izumiya et al., xe2x80x9cPeptide Synthesisxe2x80x9d, Maruzen Inc. (1975)). These peptide synthesis methods can be carried out either by a solid phase synthesis or a liquid phase synthesis.
Examples of pharmaceutically acceptable salts of peptide which is represented by the formula (I) include sodium or hydrochloride salts.
As the pharmaceutical composition of the present invention, peptides represented by the formula (1) or pharmaceutically acceptable salts thereof may directly be used as they are, or alternatively they may be formulated in combination with pharmaceutically acceptable carriers.
Examples of pharmaceutically acceptable carriers include excipients such as fillers, extenders, binders, humectants, disintegrating agents and surfactants; or diluents, which are commonly used for preparing formulations depending on usage forms. The dosage form of the pharmaceutical composition of the present invention can include, but not limited to, e.g. tablets, powders, granules, and pills, or solutions, suspensions and emulsions, so far as it effectively contains a peptide represented by the formula (I). Furthermore, the pharmaceutical composition of the present invention may be dry forms capable of changing into a liquid form by addition of a proper carrier before use. The formulation can be carried out in a usual manner.
Dose of the pharmaceutical composition of the present invention is appropriately selected depending on administration methods, dosage forms, or conditions of a patient to be administered, or the like. Generally, it is appropriate to prepare a formulation comprising a peptide of the invention at a rate of about 1-100% by weight, and to administer the peptide contained in the formulation in a dose of about 0.2-300 mg per day per adult person. Furthermore, the administration is not necessarily given once a day, but can be divided over several times a day.
The pharmaceutical composition of the invention can be administered via an administration route which depends on dosage forms. For example, in the case of an injection form, administration can be intravenous, intramuscular, subcutaneous, intracutaneous, or intraperitoneal, and in the case of a solid form, administration can be done perorally, etc.
The pharmaceutical composition of the invention has food and water intake inhibitory effects, as well as weight-reducing and obesity-inhibiting effects. Hence, the administration of the pharmaceutical composition of the invention results in food and water intake inhibitory effects, and accompanying weight-reducing and obesity-inhibiting effects, indicating that the composition is particularly useful for diseases such as obesity.
The specification incorporates the descriptions in the specification of Japanese Patent Application No. 10-172325 on which the priority of the present application is based.